Gels for encapsulation of biological materials

ABSTRACT

This invention provides novel methods for the formation of biocompatible membranes around biological materials using photopolymerization of water soluble molecules. The membranes can be used as a covering to encapsulate biological materials or biomedical devices, as a “glue” to cause more than one biological substance to adhere together, or as carriers for biologically active species. Several methods for forming these membranes are provided. Each of these methods utilizes a polymerization system containing water-soluble macromers, species, which are at once polymers and macromolecules capable of further polymerization. The macromers are polymerized using a photoinitiator (such as a dye), optionally a cocatalyst, optionally an accelerator, and radiation in the form of visible or long wavelength UV light. The reaction occurs either by suspension polymerization or by interfacial polymerization. The polymer membrane can be formed directly on the surface of the biological material, or it can be formed on material, which is already encapsulated.

“This application is a continuation of application Ser. No. 08/783,387,filed Jan. 13, 1997, now U.S. Pat. No. 6,258,870; which is a division ofSer. No. 08/484,160, filed Jun. 7, 1995, now abandoned; which is adivision of Ser. No. 07/958,870, filed Oct. 7, 1992, now U.S. Pat. No.5,529,914; which is a continuation-in-part of application Ser. No.07/870,540, filed on Apr. 20, 1992, now abandoned; which is acontinuation-in-part of application Ser. No. 07/843,485, filed on Feb.28, 1992, now abandoned. Said application Ser. No. 07/958,870, filedOct. 7, 1992, now U.S. Pat. No. 5,529,914, is also acontinuation-in-part of U.S. application Ser. No. 07/598,880, filed Oct.15, 1990, now abandoned and a continuation-in-part of U.S. applicationSer. No. 07/740,703, filed Aug. 5, 1991, now U.S. Pat. No. 5,380,536.All of the above-identified patents and applications are incorporatedherein by reference.”

BACKGROUND

Microencapsulation technology holds promise in many areas of medicine.For example, some important applications are treatment of diabetes(Goosen, et al., 1985), production of biologically important chemicals(Ornata, et al., 1979), evaluation of anti-human immunodeficiency virusdrugs (McMahon, et al., 1990), encapsulation of hemoglobin for red bloodcell substitutes, and controlled release of drugs. During encapsulationusing prior methods, cells are often exposed to processing conditions,which are potentially cytotoxic. These conditions include heat, organicsolvents and non-physiological pH, which can kill or functionally impaircells. Proteins are often exposed to conditions that are potentiallydenaturing and can result in loss of biological activity.

Further, even if cells survive processing conditions, the stringentrequirements of encapsulating polymers for biocompatibility, chemicalstability, immunoprotection and resistance to cellular overgrowth,restrict the applicability of prior art methods. For example, theencapsulating method based on ionic crosslinking of alginate (apolyanion) with polylysine or polyomithine (polycation) (Goosen, et al.,1985) offers relatively mild encapsulating conditions, but the long-termmechanical and chemical stability of such ionically crosslinked polymersremains doubtful. Moreover, these polymers when implanted in vivo, aresusceptible to cellular overgrowth (McMahon, et al., 1990), whichrestricts the permeability of the microcapsule to nutrients,metabolites, and transport proteins from the surroundings. This has beenseen to possibly lead to starvation and death of encapsulated islets ofLangerhans cells (O'Shea et al., 1986).

Thus, there is a need for a relatively mild cell encapsulation method,which offers control over properties of the encapsulating polymer. Themembranes must be non-toxically produced in the presence of cells, withthe qualities of being permselective, chemically stable, and very highlybiocompatible. A similar need exists for the encapsulation of biologicalmaterials other than cells and tissues.

Biocompatibility

Synthetic or natural materials intended to come in contact withbiological fluids or tissues are broadly classified as biomaterials.These biomaterials are considered biocompatible if they produce aminimal or no adverse response in the body. For many uses ofbiomaterials, it is desirable that the interaction between thephysiological environment and the material be minimized. For these uses,the material is considered “biocompatible” if there is minimal cellulargrowth on its surface subsequent to implantation, minimal inflammatoryreaction, and no evidence of anaphylaxis during use. Thus, the materialshould elicit neither a specific humoral nor cellular immune response,nor a nonspecific foreign body response.

Materials successful in preventing all of the above responses arerelatively rare; biocompatibility is more a matter of degree rather thanan absolute state. The first event occurring at the interface of anyimplant with surrounding biological fluids is protein adsorption(Andrade, et al., 1986). In the case of materials of natural origin, itis conceivable that specific antibodies for that material exist in therepertoire of the immune defense mechanism of the host. In this case astrong immune response can result. Most synthetic materials, however, donot elicit such a reaction. They can either activate the complementcascade or adsorb serum proteins that mediate cell adhesion, called celladhesion molecules (CAMs) (Buck, et al., 1987). The CAM family includesproteins such as fibronectin, vitronectin, laminin, von Willebrandfactor, and thrombospondin.

Proteins can adsorb on almost any type of material. They have positivelyand/or negatively charged regions, as well as hydrophilic andhydrophobic regions. They can thus interact with implanted materialthrough any of these various regions, resulting in cellularproliferation at the implant surface. Complement fragments such as C3bcan be immobilized on the implant surface and act as chemoattractants.They in turn can activate inflammatory cells such as macrophages andneutrophils and cause their adherence and activation on the implant.Those cells attempt to degrade and digest the foreign material.

In the event that the implant is nondegradable and is too large to beingested by large single activated macrophages, the inflammatory cellsmay undergo frustrated phagocytosis. Several such cells can combine toform foreign body giant cells. In this process, these cells releaseperoxides, hydrolytic enzymes, and chemoattractant and anaphylacticagents such as interleukins, which increase the severity of thereaction. They also induce the proliferation of fibroblasts on foreignsurfaces.

Fibroblasts secrets a collagenous matrix which ultimately results inencasement of the entire implant in a fibrous envelope. Cell adhesioncan also be mediated on a charged surface by the cell surfaceproteoglycans such as heparin sulfate and chondroitin sulfate (vanWachem, et al., 1987). In such a process, intermediary CAMs are notrequired and the cell surface can interact directly with the surface ofthe implant.

Enhancing Biocompatibility

Past approaches to enhancing biocompatibility of materials started withattempts at minimization of interfacial energy between the material andits aqueous surroundings. Similar interfacial tensions of the solid andliquid were expected to minimize the driving force for proteinadsorption and this was expected to lead to reduced cell adhesion andthrombogenicity of the surface. For example, Amudeshwari et al. usedcollagen gels cross-linked in the presence of hydroxyethyl methacrylate(HEMA) and methyl methacrylate (MMA) (Amudeshwari, et al., 1986). Desaiand Hubbell showed a poly (HEMA)-MMA copolymer to be somewhatnon-thrombogenic (Desai, N. P. and Hubbell, 1989).

Protein adsorption and desorption, however, is a dynamic phenomenon, asseen in the Vroman effect. This effect is the gradual displacement ofone serum protein by another, through a well-defined series, until onlyvirtually irreversibly adsorbed proteins are present on the surface.Affinity of protein in a partially dehydrated state for the polymersurface has been proposed as a determining factor for protein adsorptiononto a surface (Baier, 1990). Enhancement of surface hydrophilicity hasresulted in mixed success; increased hydrophilicity or hydrophobicitydoes not have a clear relation with biocompatibility (Coleman, et al.,1982; Hattori, et al., 1985). In some cases, surfaces with intermediatehydrophilicities demonstrate proportionately less protein adsorption.The minimization of protein adsorption may depend both uponhydrophilicity and the absence of change, as described further below,perhaps in addition to other factors.

Use of Gels in Biomaterials

Gels made of polymers which swell in water such as poly (HEMA),water-insoluble polyacrylates, and agarose, have been shown to becapable of encapsulating islet cells and other animal tissue (Iwata, etal., 1989; Lamberti, et al., 1984). However, these gels have undesirablemechanical properties. Agarose forms a weak gel, and the polyacrylatesmust be precipitated from organic solvents, thus increasing thepotential for cytotoxicity. (Dupuy et al., 1988) have reported themicroencapsulation of islets by polymerization of acrylamide to formpolyacrylamide gels. However, the polymerization process, if allowed toproceed rapidly to completion, generates local heat and requires thepresence of toxic cross-linkers. This usually results in mechanicallyweak gels whose immunoprotective ability has not been established.Moreover, the presence of a low molecular weight monomer is requiredwhich itself is cytotoxic.

Microcapsules formed by the coacervation of alginate and poly (L-lysine)(PLL) have been shown to be imnmunoprotective (O'Shea et al., 1986).However, implantation for periods up to a week has resulted in severefibrous overgrowth on these microcapsules (McMahon, et al. 1990; O'Shea,et al., 1986).

Use of Poly(ethylene oxide) (PEO) in Biomaterials

The use of poly(ethylene oxide) (PEO) to increase biocompatibility iswell-documented in the literature. The presence of grafted PEO on thesurface of bovine serum albumin has been shown by Abuchowski et al.(1977) to reduce immunogenicity in a rabbit and to increase circulationtimes of exogenous proteins in animals. The biocompatibility ofalgin-poly(L-lysine) microcapsules has been significantly enhanced byincorporating a graft copolymer of poly (L-lysine) (PLL) and PEO on themicrocapsule surface (Sawhney, et al.)

The grafting of methoxy PEO onto polyacrylonitrile surfaces was seen byMiyama et al. (1988) to render the polyacrylonitrile surface relativelynon-thrombogenic. Nagoaka et al. synthesized a graft copolymer ofmethacrylates with PEO and found the resulting polymer to be highlynon-thrombogenic. Desai and Hubbell have immobilized PEO onpoly(ethylene terepthalate) surfaces by forming a physicalinterpenetrating network (Desai et al., 1992); they have shown thesesurface to be highly resistant to thrombosis (Desai et al, 1991) and toboth mammalian and bacterial cell growth (Desai, et al.).

PEO is a unique polymer in terms of structure. The PEO chain is highlywater soluble and highly flexible. Polymethylene glycol, on the otherhand, undergoes rapid hydrolysis, while polypropylene oxide is insolublein water. PEO chains have an extremely high motility in water and arecompletely non-ionic in structure. The synthesis and characterization ofPEO derivatives which can be used for attachment of PEO to varioussurfaces, proteins, drugs etc. has been reviewed (Harris, 1985). Otherpolymers are also water soluble and non-ionic, such as poly(N-vinylpyrrolidinone) and poly(ethyl oxazoline). These have been used to reduceinteraction of cells with tissues. (Desai et al., 1991). Water solubleionic polymers, such as hyaluronic acid, have also been used to reducecell adhesion to surfaces and can similarly be used.

Immobilization of PEO on a charged surface, such as a coacervatedmembrane of alginate-PLL, results in shielding of surface charges by thenon-ionic PEO (Sawhney et al.,). The highly motile PEO chain sweeps outa free volume in its microenvironment. The free volume exclusion effectmakes the approach of a macromolecule (viz., a protein) close to asurface which has grafted PEO chains sterically unfavorable (Miyama, etal., 1988; Nagoaka, et al.; Desai, et al.; Sun, et al., 1987). Thusprotein adsorption is minimized and cell adhesion is reduced, resultingin surfaces showing increased biocompatibility.

Imobilization of PEO on a surface has been largely carried out by thesynthesis of graft copolymers having PEO side chains (Sawhney, et al.;Miyama, at al., 1988; Nagoaka et al.). This process involves the customsynthesis of monomers and polymers for each application. The use ofgraft copolymers, however, still does not guarantee that the surface“seen” by a macromolecule consists entirely of PEO.

Electron beam cross-linking has been used to synthesize PEO hydrogels,and these biomaterials have been reported to be non-thrombogenic (Sun,et al., 1987; Dennison, 1986). However, use of an electron beanprecludes the presence of any living tissue due to the sterilizingeffect of this radiation. Also, the networks produced are difficult tocharacterize due to he non-specific cross-linking induced by theelectron beam.

Photopolymerizable polyethylene glycol diacrylates have been used toentrap yeast cells for fermentation and chemical conversion (Kimura etal. 1981; Omata at al., 1981; Okada et al. 1987). However, yeast cellsare widely known to be much hardier, resistant to adverse environmentsand elevated temperatures, and more difficult to kill when compared tomammalian cells and human tissues. For example, yeast may be grownanaerobically, whereas mammalian cells may not; yeast are more resistantto organic solvents (e.g., ethanol to 12%) than are mammalian cells(e.g., ethanol to<1%); and yeast possess a polysaccharide cell wall,whereas mammalian cells, proteins, polysaccharides, and drugs do not.None of these references, however, discuss the exposure of sensitiveeukaryotic tissue, organisms, or sensitive molecules to the chemicalconditions used during polymerization because their polymerizationconditions are incompatible with sensitive materials. For example, thereare no reports of the encapsulation of mammalian cells using prior artphotosensitive prepolymers without a marked loss of cellular function.

Other earlier encapsulations of cells within photopolymerizablematerials have focused on microbial cells (Kimura et al., 1981; Omata etal., 1981; Okada et al., 1987; Tanaka et al., 1977; Omata et al.; 1979;Chun et al. 1981; Fukui et al., 1976; Fukui et al., 1984). Each of thesereports, however, describes the use of near ultraviolet light(wavelength<320 nm), which is injurious to more sensitive cells such asmammalian cells or higher eukaryotic cells. In the original presentationof the technique (Fukui et al., 1976), the authors state in the finalsentence that the technique would be appropriate for microbial cells,but provide no indication of usefulness for more sensitive cells. In amore recent and complete review of the technique (Fukui et al., 1984),the authors, in section 6 entitled “Entrapped Living Cells” provide noteaching regarding cells other than microbial cells, and in section 7entitled “Future Prospects,” they also provide no such teaching.

Moreover, the prior use of such materials for the entrapment ofbiological materials is entirely focused on industrial technology,rather than biomedical technology. For example, no attention is paid tobiocompatibility, including formulation of the gel to avoid the problemsdescribed above. This is an important issue, since bioincompatibility inbiomedical applications leads to xenograft failure in therapeuticallytransplanted cells for the evaluation of drug efficacy (O'Shea at al.,1986) and to xenograft failure in diagnostically transplanted cells(McMahon et al., 1990). Similarly, bioincompatibility would lead to thefailure of encapsulated enzymes (for example, therapeutic enzymesencapsulated and circulating or implanted in a blood-rich tissue). Suchencapsulated and entrapped enzymes could leave the circulation byinteraction with the reticuloendothelial system (Hunt et al., 1985) orcould become overgrown with tissues in a foreign body reaction.

Other ways of producing PEO hydrogels include use of PEO chains endcapped with n-alkane chains, which associate in aqueous media to formstable gels (Knowles, et al., 1990). No biological properties of thesematerials have boon reported, however. Thus, the prior art contains nodescription of methods to form biocompatible PEO networks onthree-dimensional living tissue surfaces without damaging encapsulatedtissue.

Among the techniques for encapsulating mammalian tissue with polymersother than PEO is a method of photopolymerizing the monomer2-hydroxyethyl methacrylate (“HEMA”) and the crosslinking agent ethyleneglycol dimethacrylate (“EGDA”) in a cylindrical mold containing thebiological material (Ronel, et al., 1981). The product of this reaction,a cylindrical gel with cells embedded throughout, is frozen and thenfinely ground into small particles. This technique, however, suffersfrom a number of disadvantages. First, because the cylindrical gel isbroken along random planes, shearing will often occur through pockets ofcells, leaving some cells exposed to the host immune system. Second,HEMA and EGDA are small cytotoxic molecules capable of penetrating thecellular membrane. Third, the resulting polymer membrane has uneven poresizes, which vary to an upper limit of 20 microns, thereby allowingtransit of immune response molecules. These drawbacks are reflected indata, which show that tissue remains viable for only 2-3 days after thisencapsulation process.

SUMMARY OF THE INVENTION

This invention provides novel methods for the formation of biocompatiblemembranes around biological materials using photopolymerization ofwater-soluble molecules. The membranes can be used as a covering toencapsulate biological materials or biomedical devices, as a “glue” tocause more than one biological substance to adhere together, or ascarriers for biologically active species.

Several methods for forming these membranes are provided. Each of thesemethods utilizes a polymerization system containing water-solublemacromers, species, which are at once polymers and macromoleculescapable of further polymerization. The macromers are polymerized using aphotoinitiator (such as a dye), optionally a cocatalyst, optionally anaccelerator, and radiation in the form of visible or long wavelength UVlight. The reaction occurs either by suspension polymerization or byinterfacial polymerization. The polymer membrane can be formed directlyon the surface of the biological material, or it can be formed onmaterial, which is already encapsulated.

Ultrathin membranes can be formed-by the methods described herein. Theseultrathin membranes allow for optimal diffusion of nutrient andbioregulator molecules across the membrane, and great flexibility in theshape of the membrane. Such thin membranes produce encapsulated materialwith optimal economy of volume. Biological material thus coated can bepacked into a relatively small space without interference from bulkymembranes.

The thickness and pore size of membranes formed can be varied. Thisvariability allows for “perm-selectivity”—membranes can be adjusted tothe desired degree of porosity, allowing only preferred molecules topermeate the membrane, while acting as a barrier against largerundesired molecules. Thus, the membranes are immunoprotective in thatthey prevent the transfer of antibodies or cells of the immune system.

When the encapsulated biological material is cellular in nature, theabsence of small monomers in the polymerization solution prevents thediffusion of toxic molecules into the cell. In this manner the presentinvention provides a polymerization system which is more biocompatiblethan any available in the prior art.

Additionally, the polymerization method utilizes short bursts of visibleor long wavelength UV light, which is nontoxic to biological material.Bioincompatible polymerization initiators employed in the prior art arealso eliminated.

According to the present invention, membrane formation occurs underphysiological conditions. Thus, no damage is done to the enclosedbiological material due to harsh pH, temperature, or ionic conditions.

Because the membrane adheres to the biological material, the membranecan be used as an adhesive to fasten more than one biological substancetogether. The macromers are polymerized in the presence of thesesubstances which are in close proximity. The membrane forms in theinterstices, effectively gluing the substances together.

Additionally, utilizing the tendency of the membrane to adhere tobiological material, a membrane can be formed around or on abiologically active substance to act as a carrier for that substance.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagrammatic representation of photoinitiation with xanthinedyes, ethyl eosin shown here.

FIG. 2A is a schematic representation of the process of dye diffusionunder laser irradiation to form a gel layer at the interface byphotopolymerization.

FIG. 2B is photograph of alginate/PLL microspheres containing isletscoated by the technique depicted in FIG. 2A.

FIG. 3 is a schematic representation of the microsphere coating processin oil.

FIG. 4 is a photograph of Islets of Langerhans encapsulated in a PEOgel.

FIG. 5 is a representation of a co-extrusion of an air flow apparatusfor use in mixing a 20% to 30% solution of a macromer with a cellsuspension and ethyl eosin and triethanolamine initiating system beforeexposure to laser light.

FIG. 6 is a photograph of spheres prepared using an air atomizationprocess as described in Example 6 with a PEG diacrylate macromer ofmolecular weight 400 Da and a +% solution in PBS, containing 0.1Mtriethanolamine as a cocatalyst and 0.5 mM ethyl eosin as aphotoinitiator.

FIG. 7A is a photograph of alginate-poly(L-lysine) microspheresexplanted after 4 days.

FIG. 7B is a photograph of alginate-poly(L-lysine) microspheres thathave been coated with PEG gel by the dye diffusion process beforeimplantation.

FIG. 8 is a graphical representation of the variation of cell countswith chemical composition of the microsphere overcoat, a-f.

FIG. 9 is a graphical representation of the permeability of PEO gels bya typical release profile for a PEO 18.5 kD gel.

FIG. 10 is a graphical representation of a release of BSA through gelsmade from 23% solutions of PEO diacrylates and tetraacrylates of 0.4 kDand 18.5 kD, respectively.

FIG. 11 a is a graphical representation of a plot of the length of thespike of gel formed by penetration of the laser beam into the gel versuslaser irradiation time.

FIG. 11B is a photograph of the spikes formed as a result of laser lightpenetration into the macromer.

FIG. 12A is a photograph of a phase contrast micrograph of the growth ofHFF cells on a typical PEG gel.

FIG. 12B is a photograph of a phase contrast micrograph of the growth ofHFF cells on a glass surface.

FIG. 13 is a photograph of microspheres explanted from mice as inExample 10.

FIGS. 14A-C. Creep curves for PEO diacrylate gels; test and recoveryloads are given below the abscissa: FIG. 14A-1K, FIG. 14B-6K and FIG.14C-18.5K.

DETAILED DESCRIPTION

By a variety of methods, this invention provides a means for creatingbiocompatible membranes of varying thickness on the surface of a varietyof biological materials. The polymerization occurs by a free-radical,reaction, causing a “macromer” with at least two ethyenicallyunsaturated moieties to form a crosslinked polymer. The components ofthis reaction are:

-   -   a photoinitiator, preferably eosin dye;    -   a “macromer,” preferably polyethlene glycol (PEG) diacrylate,        m.w. 18.5 kD.        This component is at once a polymer and a macromer    -   optionally a cocatalyst, preferably triethanolamine; and    -   optionally, an accelerator.

These components are mixed in varying combinations, and the mixture isexposed to longwave UV or visible light (“radiation”), preferably ofwavelength 350-700 nm, most preferred at 365-514 nm, to initiatepolymerization. A network is formed as the macromers polymerize in avariety of directions.

Four methods are used to effect polymerization to form biocompatiblemembranes. These are referred to below as the “bulk suspensionpolymerization” method, the “microcapsule suspension polymerization”method, the “microcapsule interfacial polymerization” method, and the“direct interfacial polymerization” method. They utilize eithersuspension or interfacial polymerization techniques on either coated oruncoated biological materials.

Bulk Suspension Polymerization Method

In this embodiment of the invention the core biological material ismixed in an aqueous macromer solution (composed of the macromer,cocatalyst and optionally an accelerator) with the photoinitiator. Smallglobular geometric structures such as spheres, ovoids, or oblongs areformed, preferably either by coextrusion of the aqueous solution withair or with a non-miscible substance such as oil, preferably mineraloil, or by agitation of the aqueous phase in contact with a non-misciblephase such as an oil phase to form small droplets. The macromer in theglobules is then polymerized when exposed to radiation. Because themacromer and initiator are confined to the globules, the structureresulting from polymerization is a capsule in which the biologicalmaterial is enclosed. This is a “suspension polymerization” whereby theentire aqueous portion of the globule polymerizes to form a thickmembrane around the cellular material.

Microcapsule Suspension Polymerization Method

This embodiment of the invention employs microencapsulated material as acore about which the macromer is polymerized in a suspensionpolymerization reaction. The biological material is first encapsulated,such as in an alginate microcapsule. The microcapsule is then mixed asin the first embodiment with the macromer solution and thephotoinitiator, and then polymerized by radiation.

This method takes advantage of the extreme hydrophilicity of PEGmacromer, and is especially suited for use with hydrogel microcapsulessuch as alginate-poly(L-lysine). The microsphere is swollen in water.When a macromer solution (with the initiating system) is forced to phaseseparate in a hydrophobic medium, such as mineral oil, the PEG macromersolution prefers to stay on the hydrophilic surface of the alginatemicrocapsule. When this suspension is irradiated, the PEG macromerundergoes polymerization and gelation, forming a thin layer ofpolymeric, water insoluble gel around the microsphere. Agarose beadshave been used in an analogous way by Gin et al. (1987) as scaffolds tocarry out polymerization of acrylamide. However, that method is limitedby potential toxicity associated with the use of a low molecular weightmonomer, as opposed to the macromeric precursors of the presentinvention.

This technique preferably involves coextrusion of the microcapsule in asolution of macromer and photoinitiator, the solution being in contactwith air or a liquid which is non-miscible with water, to form dropletswhich fall to a container such as a petri dish containing a solutionsuch as mineral oil in which the droplets are not miscible. Thenon-miscible liquid is chosen for its ability to maintain dropletformation. Additionally, if the membrane-encapsulated material is to beinjected or implanted in an animal, any residue should be non-toxic andnon-immunogenic. Mineral oil is a preferred non-miscible liquid.

On the petri dish the droplets are exposed to radiation which causespolymerization. This coextrusion technique results in a crosslinkedpolymer coat of greater than 50 microns thickness. Alternatively, themicrocapsules may be suspended in a solution of macromer andphotoinitiator, which is agitated in contact with a non-miscible phasesuch as an oil phase. The emulsion, which results is irradiated to forma polymer coat, again of greater than 50 microns thickness.

Microcapsule Interfacial Polymerization Method

In this embodiment, the biological material is also microencapsulated asin the previous method. However, rather than suspension polymerization,interfacial polymerization is utilized to form the membrane. Thisinvolves coating the microcapsule with photoinitiator, suspending themicrocapsule in the macromer solution, and immediately irradiating. Bythis technique a thin polymer coat, of less than 50 microns thickness,is formed about the microcapsule, because the photoinitiator is presentonly at the microcapsule surface and is given insufficient time todiffuse far into the macromer solution. As a result, the initiator ispresent in only a thin shell of the aqueous solution, causing a thinlayer to be polymerized.

When the microcapsules are in contact with dye solution, the dyepenetrates into the inner core of the microcapsule as well as adsorbingto the surface. When such a microcapsule is put into a solutioncontaining a macromer and, optionally, a cocatalyst such astriethanolamine, and exposed to laser light, initially all the essentialcomponents of the reaction are present only at and just inside theinterface of microcapsule and macromer solution. Hence, thepolymerization and gelation (if multifunctional macromer is used)initially takes place only at the interface, just beneath it, and justbeyond it. If left for longer periods of time, the dye starts diffusingfrom the inner core of the microsphere into the solution; similarly,macromers start diffusing inside the core.

Polymerization and subsequent gelation are very rapid (typical gelationtimes are 100 ms) (Fouassier, at al., 1985; Chesneau, et al., 1985).Because diffusion is a much slower process than polymerization, not theentire macromer solution is polymerized or gelled. Essentially thereaction is restricted to the near surface only. The dye, being asmaller molecule and being weakly bound to the capsule materials, keepsdiffusing out of the microsphere. If this diffusion occurs under laserirradiation, then dye at the interface is used continuously to form athicker gel layer. The thickness of the coating can thus be directed bycontrolling the reaction conditions.

A schematic representation of this process is shown in FIG. 2A. Theamount, thickness or size and rigidity of the gel formed will depend onthe size and intensity of the beam, time of exposure, initiator,macromer molecular weight, and macromer concentration (see below).Alginate/PLL microspheres containing islets coated by this technique areshown in FIG. 2B.

Direct Interfacial Polymerization Method

The fourth embodiment of this invention utilizes interfacialpolymerization to form a membrane directly on the surface of thebiological material. This results in the smallest capsules and thusachieves optimal economy of volume. Tissue is directly coated withphotoinitiator, emersed in the macromer solution, and immediatelyirradiated. This technique results in a thin polymer coat surroundingthe tissue since there is no space taken up by a microcapsule, and thephotoinitiator is again present only in a thin shell of the macromersolution.

Use as an Adhesive

It is usually difficult to get good adhesion between polymers of greatlydifferent physicochemical properties. The concept of a surface physicalinterpenetrating network was presented by Desai and Hubbel (N. P. Desaiet al. (1992)). This approach to incorporating into the surface of onepolymer a complete coating of a polymer of considerably differentproperties involved swelling the surface of the polymer to be modified(base polymer) in a mutual solvent, or a swelling solvent, for the basepolymer and for the polymer to be incorporated (penetrant polymer). Thepenetrant polymer diffused into the surface of the base polymer. Thisinterface was stabilized by rapidly precipitating or deswelling thesurface by placing the bass polymer in a nonsolvent bath. This resultedin entanglement of the penetrant polymer within the matrix of the basepolymer at its surface in a structure that was called a surface physicalinterpenetrating network.

This approach can be improved upon by photopolymerizing the penetrantpolymer upon the surface of the base polymer in the swollen state. Thisresults in much enhanced stability over that of the previous approachand in the enhancement of biological responses to these materials. Thepenetrant may be chemically modified to be a prepolymer (macromer), i.e.capable of being polymerized itself. This polymerization can beinitiated thermally or by exposure to visible, ultraviolet, infrared,gamma ray, or electron beam irradiation, or to plasma conditions. In thecase of the relatively nonspecific gamma ray or electron beam radiationreaction, chemical incorporation of particularly reactive sites may notbe necessary.

Polyethylene glycol (PEG) is a particularly useful penetrant polymer forbiomedical applications where the lack of cell adhesion is desired. Theprevious work had demonstrated an optimal performance at a molecularweight of 18,500 D without chemical crosslinking. PEG prepolymers can bereadily formed by acrylation of the hydroxyl groups at its termini orelsewhere within the chain. These prepolymers can be readily polymerizedby the above described radiation methods. Photoinititated polymerizationof these propolymers is particularly convenient and rapid. There are avariety of visible light initiated and ultraviolet light initiatedreactions that are initiated by light absorption by specificphotochemically reactive dyes, described elsewhere herein. This sameapproach can be used for biomedical purposes with other water-solublepolymers, such as poly(N-vinyl pyrrolidinone), poly(N-isopropylacrylamide), poly(ethyl oxazoline) and many others.

Additionally, it is usually difficult to obtain adhesives for wetsurfaces and tissues. Water-soluble prepolymers, for example PEGdiacrylates, can be used for this purpose. When a water-soluble polymeris placed in aqueous solution upon a tissue, the polymer diffuses intothe surface of the tissue, within the protein and polysaccharide matrixupon the tissue but not within the cells themselves. When thewater-soluble polymer is a prepolymer and a visible, ultraviolet orinfrared photoinitiator is included, the polymer penetrant may beexposed to the appropriate light to gel the polymer. In this way, thepolymer is crosslinked within and around the matrix of the tissue inwhat is called an interpenetrating network. If the prepolymer is placedin contact with two tissues and the prepolymer is illuminated, thenthese two tissues are adhered together by the intermediate polymer gel.

Biological Materials

Due to the biocompatibility of the materials and techniques involved, awide variety of materials can be used in conjunction with the presentinvention. For encapsulation, the techniques can be used with mammaliantissue and/or cells, as well as sub-cellular organelles and otherisolated sub-cellular components. The membranes can be crafted to mostthe perm-selectivity needs of the biological material enclosed. Cellsthat can [which are to] be used to produce desired products such asproteins are optimally encapsulated by this invention.

Examples of cells that [which] can be encapsulated are primary culturesas well as established cell lines, including transformed cells. Theseinclude but are not limited to pancreatic islet cells, human foreskinfibroblasts, Chinese hamster ovary cells, beta cell insulomas,lymphoblastic leukemia cells, mouse 3T3 fibroblasts, dopamine secretingventral mesencephalon cells, neuroblastoid cells, adrenal medulla cells,and T-cells. As can be seen from this partial list, cells of all types,including dermal, neural, blood, organ, muscle, glandular, reproductive,and immune system cells can be encapsulated successfully by this method.Additionally, proteins (such as hemoglobin), polysaccharides,oligonucleotides, enzymes (such as adenosine deaminase), enzyme systems,bacteria, microbes, vitamins, cofactors, blood clotting factors, drugs(such as TPA, streptokinase or heparin), antigens for immunization,hormones, and retroviruses for gene therapy can be encapsulated by thesetechniques.

The biological material can be first enclosed in a structure such as apolysaccharide gel. (Lim, U.S. Pat. No. 4,352,883; Lim, U.S. Pat. No.4,391,909; Lim, U.S. Pat. No. 4,409,331; Tsang, at al., U.S. Pat. No.4,663,286; Goosen at al., U.S. Pat. No. 4,673,556; Goosen et al., U.S.Pat. No. 4,689,293; Goosen et al., U.S. Pat. No. 4,806,355; Rha et al.,U.S. Pat. No. 4,744,933; Rha et al., U.S. Pat. No. 4,749,620,incorporated herein by reference.) Such gels can provide additionalstructural protection to the material, as well as a secondary level ofperm-selectivity.

Macromers

Polymerization via this invention utilizes macromers rather thanmonomers as the building blocks. The macromers are small polymers, whichare susceptible to polymerization into the larger polymer membranes ofthis invention. Polymerization is enabled because the macromers containcarbon-carbon double bond moieties, such as acrylate, methacrylate,ethacrylate, 2-phenyl acrylate, 2-chloro acrylate, 2-bromo acrylate,itaconate, acrylamide, methacrylamide, and styrene groups. The macromersare water soluble compounds and are non-toxic to biological materialbefore and after polymerization.

Examples of macromers are ethylenically unsaturated derivatives ofpoly(ethylene oxide) (PEO), poly(ethylene glycol) (PEG), poly(vinylalcohol) (PVA), poly(vinylpyrrolidone) (PVP), poly(thyloxazoline)(PEOX), poly(amino acids), polysaccharides such as alginate, hyaluronicacid, chondroitin sulfate, dextran, dextran sulfate, heparin, heparinsulfate, heparan sulfate, chitosan, gellan gum, xanthan gum, guar gum,water soluble cellulose derivatives and carrageenan, and proteins suchas gelatin, collagen and albumin. An example of a macromer is

where F₁=CONH, COO or NHCOO

-   -   X=H, CH₃, C₂H₅, C₆H₅,Cl, Br, OH or CH₂COOH    -   F₂=COO, CONH, O or C₆H₄,    -   R=CH₂ or -alkyl-,    -   n 5, and    -   m 3.

These macromers can vary in molecular weight from 0.2-100 kD, dependingon the use. The degree of polymerization, and the size of the startingmacromers, directly affect the porosity of the resulting membrane. Thus,the size of the macromers is [are] selected according to thepermeability needs of the membrane. For purposes of encapsulating cellsand tissue in a manner which prevents the passage of antibodies acrossthe membrane but allows passage of nutrients essential for cellularmetabolism, the preferred starting macromer size is in the range of 10kD to 18.5 kD, with the most preferred being around 18.5 kD. Smallermacromers result in polymer membranes of a higher density with smallerpores.

Photoinitiating Dyes

The photoinitiating dyes capture light energy and initiatepolymerization of the macromers. Any dye can be used which absorbs lighthaving frequency between 320 nm and 900 nm, can form free radicals, isat least partially water soluble, and is non-toxic to the biologicalmaterial at the concentration used for polymerization. Examples ofsuitable dyes are ethyl eosin, eosin Y, fluorescein, 2,2-dimethoxy,2-phenylacetophenone, 2-methoxy, 2-phenylacetophenono, camphorquinone,rose bengal, methylene blue, erythrosin, phloxime, thionine, riboflavinand methylene green. The preferred initiator dye is ethyl eosin due toits spectral properties in aqueous solution. FIG. 1 shows adiagramnnatic representation of photoinitiation with ethyl eosin.

Cocatalyst

The cocatalyst is a nitrogen based compound capable of stimulating thefree radical reaction. Primary, secondary, tertiary or quaternary aminesare suitable cocatalysts, as are any nitrogen atom containingelectron-rich molecules. Cocatalysts include, but are not limited to,triethanolamine, triethylamine, ethanolamino, N-methyl diethanolamine,N,Ndimethyl benzylamine, dibenzyl amino, N-benzyl ethanolamine,N-isopropyl benzylamino, tetramethyl ethylenediamine, potassiumpersulfate, tetramethyl ethylenediamine, lysine, omithine, histidine andarginine.

Radation Wavelength

The radiation used to initiate the polymerization is either longwave UVor visible light, with a wavelength in the range of 320-900 nm.Preferably, light in the range of 350-700 nm, and even more preferred inthe range of 365-514 nm, is used. This light can be provided by anyappropriate source able to generate the desired radiation, such as amercury lamp, longwave UV lamp, He—Ne laser, or an argon ion laser.

Thickness and Conformation of Polymer Layer

Membrane thickness affects a variety of parameters, includingperm-selectivity, rigidity, and size of the membrane. In the interfacialpolymerization method, the duration of the radiation can be varied toadjust the thickness of the polymer membrane formed. This correlationbetween membrane thickness and duration of irradiation occurs becausethe photoinitiator diffuses at a steady rate, with diffusion being acontinuously occurring process. Thus, the longer the duration ofirradiation, the more photoinitiator will initiate polymerization in themacromer mix, the more macromer will polymerize, and a thicker coat willbe formed. Additional factors that [which] affect membrane thickness arethe number of reactive groups per macromer, the concentration ofaccelerators in the macromer solution. This technique allows thecreation of very thin membranes because the photoinitiator is firstpresent in a very thin layer at the surface of the biological material,and polymerization only occurs where the photoinitiator is present.

The suspension polymerization method forms a somewhat thicker membranethan the interfacial polymerization method. This is becausepolymerization occurs in the suspension method throughout the macromermix. The thickness of membranes formed by the suspension method isdetermined in part by the viscosity of the macromer solution, theconcentration of the macromer in that solution, the fluid mechanicalenvironment of the suspension and surface active agents in thesuspension. These membranes vary in thickness from 50-300 microns. Theshape of the structure formed by suspension polymerization can becontrolled by shaping the reaction mix prior to polymerization. Spherescan be formed by emulsion with a non-miscible liquid such as oil,coextrusion with such a liquid, or coextrusion with air. Cylinders maybe formed by casting or extrusion, and slabs and discoidal shapes can beformed by casting. Additionally, the shape may be formed in relationshipto an internal supporting structure such as a screening network ofstable polymers (e.g. an alginate gel or a woven polymer fiber) ornontoxic metals.

The overall amount, thickness, and rigidity of the membrane formeddepends on the interaction of several parameters, including the size andintensity of the radiation beam, duration of exposure of the solution tothe radiation, reactivity of the initiator selected, macromer molecularweight, and macromer concentration.

The invention can be used for a variety of purposes, some of which areenumerated below, along with benefits which accrue from the use; of theinvention:

-   -   a. Microencapsulating cells: more biocompatible, stronger, more        stable, better control of permselectivity, less toxic conditions    -   b. Macroencapsulating cells: more biocompatible, stronger, more        stable, better control of permselectivity, less toxic        conditions, easier to incorporate internal or external        supporting structure    -   c. Microencapsulating or macroencapsulating other tissues, with        the same benefits    -   d. Microencapsulating or macroencapsulating pharmaceuticals:        more biocompatible, less damaging to the pharmaceutical    -   e. Coating devices: ease of application, more biocompatible    -   f. Coating microcapsules: more biocompatible, strengthens them,        ease of coating    -   g. Coating macrocapsules, microcapsules, microspheres and        macrospheres: more biocompatible, ease of coating    -   h. Coating tissues to alter adhesion of other tissues: ease of        coating, less toxicity to the tissues, conformal coating versus        nonconformal    -   i. Adhesive between two tissues: ease of adhesion, rapidity of        forming adhesive bond, loss toxicity to tissues

The invention described herein is further exemplified in the followingExamples. While these Examples provide a variety of combinations usefulin performing the methods of the invention, they are illustrative onlyand are not to be viewed as limiting in any manner the scope of theinvention.

-   -   Example 1—Synthesis of PEG 6 kD Diacrylate    -   Example 2—Synthesis of PEG 18.4 kD Tetraacrylate    -   Example 3—Coating of Islet-Containing Alginate-PLL Microspheres        by Surface Dye Adsorption    -   Example 4—Coating Islet-Containing Alginate-PLL Microspheres by        the Oil Suspension Method    -   Example 5—Encapsulation of Islets of Langerhans    -   Example 6—Microencapsulation of Animal Cells    -   Example 7—Coating of Animal Cell-Containing Alginate-PLL        Microspheres and Individual Calls by Surface Dye Adsorption    -   Example 8—Coating Animal Cell Containing Alginate-PLL        Microspheres by the Oil Suspension Method    -   Example 9—Coating of Individual Islets of Langerhans by Surface        Dye Adsorption    -   Example 10—Biocompatibility of PEO on Microspheres    -   Example 11—Permeability of PEO Gels    -   Example 12—Treatment of Silicone Rubber    -   Example 13—Treatment of Polyurethane    -   Example 14—Treatment of Ultrafiltration Membranes    -   Example 15—Treatment of Textured Materials and Hydrogels    -   Example 16—Treatment of Dense Materials    -   Example 17—Rate of Polymerization    -   Example 18—PEO Gel Interactions    -   Example 19—Characterization and Mechanical Analysis of PEO Gels    -   Example 20—Water Content of PEO Gels    -   Example 21—Mechanical Stability of PEO Gels after Implantation    -   Example 22—Monitoring of Calcification of PEO Gels    -   Example 23—Encapsulation of Neurotmrismitter-Releasing Cells    -   Example 24—Encapsulation of Hemoglobin for Synthetic        Erythrocytes    -   Example 25—Entrapment of Enzymes for Correction of Metabolic        Disorders and Chemotherapy    -   Example 26—Cellular Microencapsulation for Evaluation of        Anti-Human Immunodeficiency Virus Drugs In Vivo    -   Example 27—Use of PEG Gels as Adhesive to Rejoin Severed Nerve    -   Example 28—Surgical Adhesive    -   Example 29—Modification of PVA Polymer    -   Example 30—Use of Alternative Photopolymerizable Moieties    -   Example 31—Use of Alternative Photoinitiator/Photosensitizer        Systems    -   Example 32—Formation of Alginate-PLL-Alginate Microcapsules with        Photopolymerizable Polycations

EXAMPLE 1 Synthesis of PRO 6 KD Diacrylate

PEG acrylates of molecular weights 400 Da and 1,000 Da ware commerciallyavailable from Sartomer and Dajac Inc., respectively. PEG 6 kD (20 g)was dissolved in 200 mL dichloromethane in a 250 mL round bottom flask.The flask was cooled to 0° C. and 1.44 mL of triethyl amino and 1.3 mLof acryloyl chloride were added with constant stirring under a drynitrogen atmosphere. The reaction mixture was then brought to roomtemperature and stirred for 12 hr under a nitrogen atmosphere. It wasthen filtered, and the filtrate was precipitated by adding to a largeexcess of hexane. The crude monomer was purified by dissolving indichloromethane and precipitating in hexane. Yield 69%.

EXAMPLE 2 Synthesis of PEG 18.4 KD Tetraacrylate

A tetrafunctional water soluble PEG (30 g; m.w. 18.5 kD) having thefollowing structure was purchased from Polysciences, Inc.:

where F=CONH, COO or NHCOO

-   -   X=H, CH₃, C₂H₅, C₆H₅, Cl, Br, OH or CH₂COOH    -   F₂=COO, CONH, O or C₆H₄, and    -   R=CH₂ or -alkyl-.

The PEG was dried by dissolving in benzene and distilling off thewater-benzene azeotrope. PEG 18.5 kD (59 g) was dissolved in 300 mL ofbenzene in a 500 mL flask. To this, 3.6 mL of triethylamine and 2.2 mLof acryloyl chloride were added under nitrogen atmosphere and thereaction mixture was refluxed for 2 hours. It was then cooled andstirred overnight. The triethyl amine hydrochloride was separated byfiltration and the copolymer was recovered from filtrate byprecipitating in a largo excess of hexane. The polymer was furtherpurified by dissolving in methylene chloride and reprecipitating inhexane. The polymer was dried at 50 degrees C under vacuum for 1 day.Yield 68%.

EXAMPLE 3 Coating of Islet-containing Alginate-PLL Microspheres bySurface Dye Adsorption

The microcapsule interfacial polymerization method was used to formmembrane around alginate-PLL microcapsules containing islets.Alginate-PLL coacervated microspheres, containing one or two humanpancreatic islets each, were suspended in a 1.1% CaCl₂ solution andaspirated free of excess solution to obtain a dense plug ofmicrospheres. A solution of ethyl eosin (0.04% w/v) was prepared in a1.1% CaCl₂ solution. This solution was filter-sterilized by passagethrough a 0.45 μm filter. The plug of microspheres was suspended in 10mL of the eosin solution for 2 min to allow uptake of the dye. Themicrospheres were then washed four times with fresh 1.1% CaCl₂ to removeexcess dye. A solution of PEG 18.5 tetraacrylate (2 mL; 23% w/v)containing 100 μL of a 3.5% w/v solution of triethanolamine in HEPESbuffered saline was added to 0.5 mL of those microspheres. Themicrospheres were exposed to argon ion laser light for 30 seconds withperiodic agitation. The suspension of microspheres was uniformly scannedwith the light during this period. The microspheres were then washedwith calcium solution and the process was repeated in order to furtherstabilize the coating.

A static glucose stimulation test (SGS) was performed on isletsencapsulated in the microspheres coated with PEG gel. Data for insulinsecretion in response to this challenge appears in Table 1. The isletswere seen to be viable by dithizone staining. The SGS test data confirmthe vitality and functionality of the islets.

TABLE 1 SGS pulse subsequent initial 300 60 Glucose Concentration (mg %)60 Insulin/Islet/hr (μU/mL)* Diffusion Overcoat Method 1.0 10.04 ± 3.562.5450.76 Mineral Oil Overcoat Method 1.0 10.23 ± 3.28 1.0250.78 FreeIslet Control 1.0 3.74 ± 1.4 1.950.17 *Values are mean ± S.D., all arenormalized as compared to the initial 60 mg %, after subjection to the300 mg % glucose, the islets were resubjected to the initial dose.

PEG diacrylate macromers may be polymerized identically as the PEGtetraacrylate macromer described in this example.

EXAMPLE 4 Coating Islet-containing Alginate-PLL Microspheres by theMicrocapsule Suspension Polymerization Method

This method takes advantage of the hydrophilic nature of PEG monomers.Alginate/PLL microspheres (2 mL), containing one or two human pancreaticislets each, were mixed with PEG tetraacrylate macromer solution (PEGmol wt 18.5 kD, 23% solution in saline) in a 50 mL transparentcentrifuge tube. Triethanolamine 0.1 M) and 0.5 mM ethyl eosin weremixed with macromer solution. The excess of macromer solution wasdecanted, 20 mL of mineral oil was added to the tube, and the reactionmixture was vortexed thoroughly for 5 minutes. Silicone oil will performequally well in this synthesis but may have poorer adjuvantcharacteristics if there is any carry-over. Any other water-immiscibleliquid may be used as the “oil” phase. Acceptable triethanolamineconcentrations range from about 1 mM to about 100 mM. Acceptable ethyleosin concentrations range from about 0.01 mM to more than 10 mM.

The beads were slightly red due to the thin coating of macromer/dyesolution, and they were irradiated for 20-50 sec with an argon ion laser(power 50-500 mW). Bleaching of the (red) ethyl eosin color suggestedcompletion of the reaction. The beads were then separated from mineraloil and washed several times with saline solution. The entire procedurewas carried out under sterile conditions.

A schematic representation of the microsphere coating process in oil isshown in FIG. 3. Alginate/polylysine capsules are soluble in sodiumcitrate at pH 12. When these coated microspheres came in contact withsodium citrate at pH 12, the inner alginate/polylysine coacervatedissolves and a PEG polymeric membrane could still be seen (crosslinkedPEG gels are substantially insoluble in all solvents including water andsodium citrate at pH 12). The uncoated control microspheres dissolvedcompletely and rapidly in the same solution.

A static glucose challenge was performed on the islets as in Example 3.Data again appear in Table 1. The islets were seen to be viable andfunctional.

EXAMPLE 5 Encapsulation of Islets of Langerhans

This example makes use of the direct interfacial polymerization. Isletsof Langerhans isolated from a human pancreas were encapsulated in PEGtetraacrylate macromer gels. 500 islets suspended in RPMI 1640 mediumcontaining 10% fetal bovine serum were pelleted by centrifuging at longfor 3 min. The pellet was resuspended in 1 mL of a 23% w/v solution ofPEO 18.5 kD diacrylate macromer in HEPES buffered saline. An ethyl eosinsolution (5 μL) in vinyl pyrrolidone (at a concentration of 0.5%) wasadded to this solution along with 100 μL of a 5 M solution oftriethanolamine in saline. Mineral oil (20 mL) was then added to thetube, which was vigorously agitated to form a dispersion of droplets200-500 μm in size. This dispersion was then exposed to an argon ionlaser with a power of 250 mW, emitting at 514 nm, for 30 sec. Themineral oil was then separated by allowing the microspheres to settle,and the resulting microspheres were washed twice with PBS, once withhexane and finally thrice with media.

FIG. 4 shows islets of Langerhans encapsulated in a PEO gel. Theviability of the islets was verified by an acridine orange and propidiumiodide staining method and also by dithizone staining. In order to testfunctional normalcy, an SGS test was performed on these islets. Theresponse of the encapsulated islets was compared to that of free isletsmaintained in culture for the same time period. All islets weremaintained in culture for a week before the SGS was performed. Theresults are summarized in Table 2. It can be seen that the encapsulatedislets secreted significantly (p<0.05) higher insulin than the freeislets. The PEO gel encapsulation process did not impair function of theislets and in fact helped then maintain their function in culture betterthan if they had not been encapsulated.

TABLE 2 Islet Insulin secretion pulse subsequent initial 300 60 GlucoseConcentration (ma %) 60 Insulin/Islet/hr (μU/mL)* Free islets 1.0  3.74+/− 1.40 1.9 +/− 0.17 Encapsulated Islets 1.0 20.81 +/− 9.36 2.0 +/−0.76 *Values are mean +/− S.D., normalized to initial basal level at 60mg % glucose.

EXAMPLE 6 Microencapsulation of Animal Cells

PEG diacrylates of different molecular weight were synthesized by areaction of acryloyl chloride with PEG as in Example 1. A 20% to 30%solution of macromer was mixed with a cell suspension and the ethyleosin and triethanolamine initiating system before exposing it to laserlight through a coextrusion air flow apparatus, FIG. 5. Microsphereswere prepared by an air atomization process in which a stream ofmacromer was atomized by an annular stream of air. The air flow rateused was 1,600 cc/min and macromer flow-rate was 0.5 mL/min. Thedroplets were allowed to fall to a petri dish containing mineral oil andwere exposed to laser light for about 0.15 sec each to causepolymerization and make then insoluble in water. Microspheres so formedwere separated from the oil and thoroughly washed with PBS buffer toremove unreacted macromer and residual initiator. The size and shape ofmicrospheres was dependent on extrusion rate (0.05 to 0.1 mL/min) andextruding capillary diameter (18 Ga to 25 Ga). The polymerization timeswere dependent on initiator concentration (ethyl eosin concentration (5μM to 0.5 mM), vinyl pyrrolidone concentration (0.0% to 0.1%),triethanolamine concentration (5 to 100 mM), laser power (10 mW to 1 W),and macromer concentration (>10% w/v).

A PEG diacrylate macromer of molecular weight 400 Da was used as a 30%solution in PBS, containing 0.1 M triethanolamine as a cocatalyst and0.5 mM ethyl eosin as a photoinitiator. Spheres prepared using thismethod are shown in FIG. 6. The polymerizations were carried out atphysiological pH in the presence of air. This is significant sinceradical polymerizations may be affected by the presence of oxygen, andthe acrylate polymerization is still rapid enough to proceedeffectively.

The process also works at lower temperatures. For cellularencapsulation, a 23% solution of PEO diacrylate was used with initiatingand polymerization conditions as used in the air atomization technique.Cell viability subsequent to encapsulation was checked by trypan blueexclusion assay. Human foreskin fibroblasts (HFF), Chinese hamster ovarycells (CHO-K1), and a beta cell insuloma line (RiN5F) were found to beviable (more than 95%) after encapsulation. A wide range (>10%) of PEGdiacrylate concentrations may be used equally effectively, as may PEGtetraacrylate macromers.

EXAMPLE 7 Coating of Animal Cell-containing Alginate-PLL Microspheresand Individual Cells by Surface Dye Adsorption

Alginate-PLL coacervated microspheres, containing animal cells, weresuspended in a 1.1% CaCl₂ solution and were aspirated free of excesssolution to obtain a dense plug of microspheres. A solution was filtersterilized by passage through a 0.45 pm filter. The plug of microsphereswas suspended in 10 mL of eosin solution for 2 min to allow dye uptake.A solution of PEG 18.5 tetraacrylate (2 mL; 23% w/v) containing 100 μLof a 3.5 w/v solution of triethanolamine in HEPES buffered saline wasadded to 0.5 mL of these microspheres. The microspheres were exposed toan argon ion laser for 30 seconds with periodic agitation. Thesuspension of microspheres was uniformly scanned with the laser duringthis period. The microspheres were then washed with calcium solution andthe process was repeated once more in order to attain a stable coating.

In order to verify survival of cells after the overcoat process, cellsin suspension without the alginate/PLL microcapsule were exposed tosimilar polymerization conditions. 1 mL of lymphoblastic leukemia cells(RAJI) (5×10⁵ cells) was centrifuged at 300 g for 3 min. A 0.04% filtersterilized ethyl eosin solution in phosphate buffered saline (PBS) (1mL) was added and the pellet was resuspended. The cells were exposed tothe dye for 1 min and washed twice with PBS and then pelleted.Triethanolamine solution (10 μL; 0.1 M) was added to the pellet and thetube was vortexed to resuspend the cells. 0.5 mL of PEO 18.5 kDtetraacrylate macromer was then mixed along with this suspension and theresulting mixture was exposed to an argon ion laser (514 nm, 50 mW) for45 sec. The cells were then washed twice with 10 mL saline and once withmedia (RPMI 1640 with 10% FCS and 1% antibiotic, antimycotic). A thinmembrane of PEO gel may be observed forming around each individual cell.

No significant difference in viability was seen between the controlpopulation (93% viable) and the treated cells (95% viable) by trypanblue exclusion. An assay for cell viability and function was performedby adapting the MTT-Formazan assay for the RAJI cells. This assayindicates >90% survival. Similar assays were performed with two othermodel cell lines. Chinese hamster ovary cells (CHO-K1) show nosignificant difference (p<0.05) in metabolic function as evaluated bythe MTT-Formazan assay. 3T3 mouse fibroblasts also show no significantreduction (p<0.05) in metabolic activity.

EXAMPLE 8 Coating Animal Cell Containing Alginate-PLL Microspheres bythe Oil Suspension Method

Using the method described in Example 4, RAJI cells contained inalginate-PLL microspheres wore coated with a PEG polymeric membrane.Viability of these cells was checked by trypan blue exclusion and theywere found to be more than 95% viable.

EXAMPLE 9 Coating of Individual Islets of Langerhans by Surface DyeAdsorption

Using the method described in Example 7, ethyl eosin was adsorbed to thesurfaces of islets, exposed to a solution of the PEG macromer withtriethanolamine, and exposed to light from an argon-ion laser to form athin PEG polymeric membrane on the surface of the islets. Isletviability was demonstrated by lack of staining with propidium iodide.

EXAMPLE 10 Biocompatability of PEO on Microspheres

In vivo evaluation of the extent of inflammatory response tomicrospheres prepared in Examples 7 and 8 was carried out byimplantation in the peritoneal cavity of mice. Approximately 0.5 mL ofmicrospheres were suspended in 5 mL of sterile HEPES buffered saline. Aportion of this suspension (2.5 mL) was injected into the peritonealcavity of ICR male Swiss white mice. The microspheres were recoveredafter 4 days by conducting a lavage of the peritoneal cavity with 5 mLof 10 U heparin/mL PBS. The extent of cellular growth on themicrospheres was visually inspected under a phase contrast microscope.The number of unattached cells present in the recovered lavage fluid wascounted using a Coulter counter. FIG. 7A shows a photograph ofalginate-poly(L-lysine) microspheres explanted after 4 days, while FIG.7B shows similar spheres which had been coated with PEG gel by the dyediffusion process before implantation. As expected, bilayeralginate-polylysine capsules not containing an outer alginate layer toprovide an extreme test of the ability of the PEG gel layer to enhancethe biocompatibility of the bilayer membrane, were completely coveredwith cells due to the highly cell adhesive nature of the PLL surface,whereas the PEG coated microspheres were virtually free of adherentcells. Almost complete coverage of alginate-poly(L-lysine) was expectedbecause polylysine has amino groups on the surface, and positivelycharged surface amines can interact with cell surface proteoglycans andsupport cell growth (Rouvony, et al., 1983). The photographs in FIG. 7Bstrongly indicate that the highly charged and cell adhesive surface ofPLL is covered by a stable layer of PEG gel. The integrity of the geldid not appear to be compromised.

The non-cell-adhesive tendency of these microspheres was evaluated as apercentage of the total microsphere area, which appears covered withcellular overgrowth. These results are summarized in Table 3.

TABLE 3 Microsphere Coverage with Cell Overgrowth Composition of PEG gel% Cell coverage 18.5 kD <1 18.5 kD 90%:0.4 kD 10% <1 18.5 kD 50%:0.4 kD50% <1 35 k 90%:0.4 kD 10% 5-7 35 k 50%:0.4 kD 50% <1 Alginate poly(L-lysine) 60-80

An increase in cell count was a result of activation of residentmacrophages, which secrete chemical factors such as interleukins andinduce nonresident macrophages to migrate to the implant site. Thefactors also attract fibroblasts responsible for collagen synthesis. Thevariation of cell counts with chemical composition of the overcoat isshown FIGS. 8(A-F). It can be seen from the figure that all PEG coatedspheres have substantially reduced cell counts. This is consistent withthe PEG overcoat generally causing no irritation of the peritonealcavity.

However, PEG composition does make a difference in biocompatibility, andincreasing molecular weights were associated with a reduction in cellcounts. This could be due to the gels made from higher molecular weightoligomers having higher potential for steric repulsion due to the longerchain lengths.

EXAMPLE 11 Permability of PEO Gels

Bovine serum albumin, human IgG, or human fibrinogen (20 mg) wasdissolved in 2 mL of a 23% w/v solution of oligomeric PEO 18.5 kDtetraacrylate in PBS. This solution was laser polymerized to produce agel 2 cm×2 cm×0.5 cm in size. The diffusion of bovine serum albumin,human IgG and human fibrinogen (mol wt 66 kD, 150 kD and 350 kDrespectively) was monitored through the 2 cm×2 cm face of these gelsusing a total protein assay reagent (Biorad). A typical release profilefor a PEO 18.5 kD gel is shown in FIG. 9. This gel allowed a slowtransport of albumin but did not allow IgG and fibrinogen to diffuse.This indicates that these gels are capable of being used asimmunoprotective barriers. This is a vital requirement for a successfulanimal tissue microencapsulation material.

The release profile was found to be a function of crosslink density andmolecular weight of the polyethylene glycol segment of the monomer. FIG.10 shows the release of BSA through gels made from 23% solutions of PEOdiacrylates and tetraacrylates of 0.4 kD and 18.5 kD, respectively. Itis evident that the 18.5 kD gel is freely permeable to albumin while the0.4 kD gel restricted the diffusion of albumin. The release of anysubstance from these gels will depend on the crosslink density of thenetwork and will also depend on the motility of the PEG segments in thenetwork. This effect is also dependent upon the functionality of themacromer. For example, the permeability of a PEG 18.5 kD tetraacrylategel is less than that of an otherwise similar PEG 20 kD diacrylate gel.

In the case of short PEO chains between crosslinks, the “pore” producedin the network will have relatively rigid boundaries and will berelatively small and so a macromolecule attempting to diffuse throughthis gel will be predominantly restricted by a sieving effect. If thechain length between crosslinks is long, the chain can fold and movearound with a high motility and, besides the sieving effect, a diffusingmacromolecule will also encounter a free volume exclusion effect.

Due to these two contrasting effects a straightforward relation betweenmolecular weight cutoff for diffusion and the molecular weight of thestarting oligomer is not completely definable. Yet, a desired releaseprofile for a particular protein or a drug such as a peptide may beaccomplished by adjusting the crosslink density and length of PEGsegments. Correspondingly, a desired protein perm ability profile may bearranged to permit the diffusion of nutrients, oxygen, carbon dioxide,waste products, hormones, growth factors, transport proteins, andreleased cellularly synthesized proteins, while restricting thediffusion of antibodies and complement proteins and also the ingress ofcells, to provide imnmunoprotectivity to transplanted cells or tissue.The three dimensional crosslinked covalently bonded polymeric network ischemically stable for long-term in vivo applications.

EXAMPLE 12 Treatment of Silicone Rubber to Enhance Biocompatibility

Pieces of medical grade silicone rubber (2×2 cm) were soaked for 1 h inbenzene containing 23% 0.4 kD PEG diacrylate and 0.5%2,2-dimethoxy-2-phenyl acetophenone. The thus swollen rubber wasirradiated for 15 min with a long wave UV lamp (365 nm). Afterirradiation, the sample was rinsed in benzene and dried. The air contactangles of silicone rubber under water were measured before and aftertreatment. The decreased contact angle of 500 after treatment, over theinitial contact angle of 630 for untreated silicone rubber reflects anincreased hydrophilicity due to the presence of the PEG gel on therubber surface.

This technique demonstrates that macromer polymerization can be used tomodify a polymer surface so as to enhance biocompatability. Forinstance, a polyurethane catheter can be treated by this method toobtain an implantable device coated with PEG. The PEG was firmlyanchored to the surface of the polyurethane catheter because themacromer was allowed to penetrate the catheter surface (to a depth of1-2 microns) during the soaking period before photopolymerization. Uponirradiation, an interpenetrating network of PEG and polyurethaneresults. The PEG was thereby inextricably intertwined with thepolyurethane.

EXAMPLE 13 Treatment of Polyurethane

INTRACATH (Becton Dickinson) polyurethane intravenous catheters (19 ga)were modified at their outer surfaces with polyethylene glycoldiacrylate (PEG DA) of molecular weight 400 and 10000. The prepolymerwas dissolved in tetrahydrofiran (THF), a solvent for the polyurethane,at 50° C., where polyurethane dissolution is relatively slow. Thefollowing solution was prepared and warmed to 50° C.:

PEG DA (MW 400) 15% PEG DA (MW 10000) 15% THF 70%with 2,2-dimethoxy, 2-phenyl acetophenone at 1.6% of the above solution.

2.5″ length catheter segments were closed at one end by melting a 2 mmlength by pressing with a hot metal spatula to from a flat tab. This tabwas used to fix the catheter in the vessel wall in subsequent animalexperiments. The catheter was held with forceps at the tab end anddipped in the treatment solution for 1-3 sec, pulled out, and the excessfluid shaken off. The treated catheter was illuminated with anultraviolet light (Black Ray, 360 nn) for 2-3 min, rotating thecatheter. An untreated control was similarly treated in 70% THF with 30%water replacing the PEG in the treatment solution.

Following this treatment, both the treated and control catheters weretransferred to 100% methylene chloride to extract unreacted materials;this extraction was carried out for 36 hr with solvent replacement every6 hr. These catheters were then dried and transferred to 70% ethanol,and then into water before use.

A second composition was also investigated:

PEG DA (MW 400) 10% PEG DA (MW 10000) 15% Polyethylene oxide (MW100,000)  5% THF 70%with 2,2-dimethoxy, 2-phenyl acetophenone at 1.6% of the above solution.

In this case, the polyethylene oxide of mw 100,000 was not a prepolymerand was immobilized within the PEG DA matrix by entanglement, ratherthan by chemical attachment.

Adult New Zealand male rabbits (7-10 lb) were anesthetized withrompun-acepromazien-ketamine. The animal was shaved on the ventrolateraljugular and the vessel was raised. A catheter was inserted into thevessel with the tab outside, and tied in place via the tab with 4.0nylon to the adventitia. The catheter was inserted 1.5 to 2.0″ into thevessel. The skin incision was closed.

After a period of 3 days, the animals were euthanized by overdose ofpentobarbital intraperitoneally. The vessel was again raised and flushedwith phosphate buffered saline (PBS) to superficially rinse away bloodbetween the catheter and the vessel wall. Two 500 ml bottles, one filledwith PBS and one with formalin in PBS were hung from an i.v. polescaffold, and the hydraulic differential was used to perfusion fix thevessel. The vessels were removed proximal and distal to the ends of thecatheters.

The treated catheters were completely wettable, and were very slippery.

A total of 12 rabbits were catheterized for 72 hr. Six were control,unmodified catheters. These catheters could not be removed from thevessel wall without dissection, i.e. they were tightly incorporated intothe vessel. These catheters upon removal were red, and the vessel wasbarely patent. By contrast, the treated catheters were easily removable,the vessels were clearly patent, and the catheters were not red. Underthe light microscope, a small amount of white thrombus could be seen onboth formulations of the catheter coating, with somewhat lesser amountson the formulation containing the polyethylene oxide 100,000.

EXAMPLE 14 Treatment of Ultrafiltration Membranes

The processes of Examples described above can be applied to thetreatment of macrocapsular surfaces, such as those used forultrafiltration, hemodialysis and non-microencapsulated immunoisolationof animal tissue. The macrocapsule in this case will usually bemicroporous with a molecular weight cutoff below 70,000 Da. It may be inthe form of a hollow fiber, a spiral module, a flat sheet or otherconfiguration. The surface of such a macrocapsule can easily be modifiedusing the PEO gel coating process to produce a nonfouling,non-thrombogenic, and non-cell-adhesive surface. The coating serves toenhance biocompatibility and to offer additional immunoprotection.Materials which can be modified in this manner include polysulfones,cellulosic membranes, polycarbonates, polyamides, polyimides,polybenzimidazoles, nylons, and poly(acrylonitrile-co-vinyl chloride)copolymers and the like.

Depending on the physical and chemical nature of the surface a varietyof methods can be employed to form biocompatible overcoats. Hydrophilicsurfaces can simply be coated by applying a thin layer of a 30% w/vpolymerizable. solution of PEG diacrylate containing appropriate amountsof dye and amine. Hydrophobic surfaces can be first rendered hydrophilicby gas plasma discharge treatment and the resulting surface can then besimilarly coated, or they may simply be treated with a surfactant beforeor during treatment with the PEG diacrylate solution.

EXAMPLE 15 Treatment of Textured Materials and Rydrogels

The surface of materials having a certain degree of surface texture,such as woven dacron, dacron velour, and expandedpoly(tetrafluoroethylene) (ePTFE) membranes, was treated using thecoating method described herein. Textured and macroporous surfaces allowgreater adhesion of the PEG gel to the material surface. This allows thecoating of relatively hydrophobic materials such as PTFE andpoly(ethylene terepthalate) (PET).

Implantable materials such as enzymatic and ion sensitive electrodes,having a hydrogel (such as poly (HEMA), crosslinked poly(vinyl alcohol)and poly(vinyl pyrrolidone)) on their surface, are coated with the morebiocompatible PEO gel in a manner similar to the dye adsorption andpolymerization technique used for the alginate-PLL microspheres.

EXAMPLE 16 Treatment of Dense Materials

The surfaces of dense (e.g., nontextured, nongel) materials such aspolymers (including PET, PTFE, polycarbonates, polyamides, polysulfones,polyurethanes, polyethylene, polypropylene, polystyrene), glass, andceramics can be treated with PEO gel coatings. Hydrophobic surfaces wereinitially treated by a gas plasma discharge to render the surfacehydrophilic. This ensures better adhesion of the PEO gel coating to thesurface. Alternatively, coupling agents may be used to increaseadhesion, as readily apparent to those skilled in the art of polymersynthesis.

EXAMPLE 17 Rate of Polymerization

To demonstrate rapidity of gelation in laser-initiated polymerizationsof multifunctional acrylic monomers, the kinetics of a typical reactionwere investigated. Trimethylolpropyl tri-acrylate containing 5×10⁻⁴ Methyl eosin as a photoinitiator in 10 μ moles of N-vinyl pyrrolidone permL of macromer mix and 0.1 M of triethanolamine as a cocatalyst, wasirradiated with a 500 mW argon ion laser (514 nm wavelength, power3.05×10⁵ W/m², beam diameter 1 mm, average gel diameter produced 1 m). Aplot of the length of the spike of gel formed by penetration of thelaser beam into the gel versus laser irradiation time is shown in FIG.11A The spikes formed as a result of laser light penetration into themacromer can be seen in FIG. 11B.

A 23% w/w solution of various macromers in HEPES buffered salinecontaining 3 μL of initiator solution (300 mg/mL of2,2-dimethoxy-2-phenylacetophenone in N-vinyl pyrrolidone) was used. 100μL of the solution was placed on a glass coverslip and irradiated with alow intensity long wave UV (LWUV) lamp (BlakRay, model 3-100A withflood). The times required for gelation to occur were noted and aregiven in Table 4. Those times were typically in the range of 10 seconds.

TABLE 4 Gelling Time Gel Time (sec) Polymer Code (mean ± S.D.) 0.4 kD6.9 ± 0.5 1 kD 21.3 ± 2.4  6 kD 14.2 ± 0.5  10 kD 8.3 ± 0.2 18.5 kD 6.9± 0.1 20 kD 9.0 ± 0.4

Time periods of about 10-100 ms were sufficient to gel a 300 μm diameterdroplet (a typical size of gel used in microencapsulation technology).This rapid gelation, if used in conjunction with proper choice ofmacromers, can load to entrapment of living cells in a three dimensionalcovalently bonded polymeric network. The monochromatic laser light willnot be absorbed by the cells unless a proper chromophore is present, andis considered to be harmless if wavelength is more than about 400 nm.Exposure to long wavelength ultraviolet light (>360 mn) is harmless atpractical intensities and durations.

EXAMPLE 18 PEO Gel Interactions Biocompatibility With HFF (HumanForeskin Fibroblasts) Cells was Demonstrated as Follows

HFF cells were seeded on PEO 18.5 kD tetraacrylate gels at a density of18,000 cells/cm² in Dulbecco's modification of Eagle's medium containing10% fetal calf serum. The gels were then incubated at 37° C. in a 5% CO₂environment for 4 hr. At the end of this time the gels were washed withPBS to remove any non-adherent cells and were observed under a phasecontrast microscope at a magnification of 200×. FIG. 12A shows thegrowth of these cells on a typical PEG gel as compared to glass surface(FIG. 12B). The number of attached cells/cm² was found to be 510±170 onthe gel surfaces as compared to 13,200±3,910 for a control glasssurface. The cells on these gels appeared rounded and were not in theirnormal spread morphology, strongly indicating that these gels do notencourage cell attachment.

Biocompatibility on microspheres was demonstrated as follows. FIG. 13shows a photograph of microspheres explanted from mice as in Example 10;after 4 days very little fibrous overgrowth was seen. The resistance ofPEG chains to protein adsorption and hence cellular growth was welldocumented. Table 5 summarizes the extent of cellular overgrowth seen onthese microspheres after 4 day intraperitoneal implants for various PEGdiacrylate gels.

TABLE 5 PEG Diacrylate for Gels (mol wt, Daltons) Extent of CellularOvergrowth 400  5-10% 1,000 15-25% 5,000  3-5% 6,000  2-15% 10,00010-20% 18,000  4-10%

EXAMPLE 19 Characterization and Mechanical Analysis of PEO Gels

Solutions of PEO diacrylates (23% w/v; 0.4 kD, 6 kD, 10 kD) and PEGtetraacrylates (18.5 kD) were used. An initiator solution (10 μL)containing 30 mg/mL of 2,2-dimethoxy-2-phenyl acetophenone invinyl-2-pyrrolidone was used per mL of the macromer solution. Thesolution of initiator containing macromer was placed in a 4.0×1.0×0.5 cmmold and exposed to a long wave ultraviolet lamp (365 nm) forapproximately 10 seconds to induce gelation. Samples were allowed toequilibrate in phosphate buffered saline (pH 7.4) for 1 week beforeanalysis 1 performed.

A series of “dogbone” samples (samples cut from a slab into the shape ofa dogbone, with wide regions at both ends and a narrower long region inthe middle) were cut for ultimate tensile strength tests. Thickness ofthe samples was defined by the thickness of the sample from which theywere cut. These thicknesses ranged from approximately 0.5 mm to 1.75 mm.The samples were 20 mm long and 2 mm wide at a narrow “neck” region. Thestress strain tests wore run in length control at a rate of 4% persecond. After each test, the cross sectional area was determined. Table6 shows the ultimate tensile strength data. It is seen that the lowermolecular weight macromers in general give stronger gels, which wereless extensible than those made using the higher molecular weightmacromers. The PEG 18.5 kD tetraacrylate gel is seen to be anomalous inthis series, resulting from the multifunctionality of the macromer andthe corresponding higher crosslinking density in the resulting gel. Thistypo of strengthening result could be similarly achieved with macromersobtained having other than four free radical sensitive groups, such asacrylate groups.

TABLE 6 Gel strength Tests PEO Acrylate Precursor Molecular Weight 0.4kD 6 kD 10 kD 18.5 kD Stress (kPa)* 168 +/− 51 98 +/− 15 33 +/− 7  115+/− 56  % Strain*  8 +/− 3 71 +/− 13 110 +/− 9  40 +/− 15 Slope* 22 +/−5 1.32 +/− 0.31 0.27 +/− 0.04 2.67 +/− 0.55 *Values are mean +/− S.D.

For the creep tests, eight samples approximately 0.2×0.4×2 cm wereloaded while submersed in saline solution. They were tested with aconstant unique predetermined load for one hour and a small recoveryload for ten minutes. Gels made from PEG diacrylates of 1 kD, 6 kD, and10 kD, and PEG tetraacrylates of 18.5 kD PEO molecular weight were usedfor this study. The 10 kD test was terminated due to a limit error (thesample stretched beyond the travel of the loading frame). The 1 kDsample was tested with a load of 10 g and a recovery load of 0.2 g. The6 kD sample was tested at a load of 13 g with a recovery load of 0.5 g.The 18.5 kD sample was tested at a load of 13 g with a recovery load of0.2 g. The choice of loads for these samples produced classical creepcurves with primary and secondary regions.

EXAMPLE 20 Water Content of PEO Gels

Solutions of various macromers were made as described above. Gels in theshape of discs were made using a mold. The solutions (400 μL) was usedfor each disc. The solutions were irradiated for 2 minutes to ensurethorough gelation. The disc shaped gels were removed and dried undervacuum at 60° C. for 2 days. The discs were weighed (WI) and thenextracted repeatedly with chloroform for 1 day. The discs were driedagain and weighed (W2). The gel fraction was calculated as W2/W1. Thisdata appears in Table 7.

Determination of Degree of Hydration

Subsequent to extraction, the discs were allowed to equilibrate with HBSfor 6 hours and weighed (W3) after excess water had been carefullyswabbed away. The total water content was calculated as (W3−W2)×100/W3.The data for gel water contents is summarized in the following table.

TABLE 7 Polymer Coat % Total Water % Gel Content 0.4 kD — 99.8 ± 1.9 1kD 79.8 ± 2.1 94.5 ± 2.0 6 kD 95.2 ± 2.5 69.4 ± 0.6 10 kD 91.4 ± 1.696.9 ± 1.5 18.5 kD 91.4 ± 0.9 80.3 ± 0.9 20 kD 94.4 ± 0.6 85.0 ± 0.4

EXAMPLE 21 Mechanical Stability of PEO Gels After Implantation

PEG diacrylate (10 kD) and PEG tetraacrylate (18.5 kD) were cast indogbone shapes as described in Example 19. PEG—dacrylate ortatraacrylate (23% w/w) in sterile HEPES buffered saline (HBS) (0.9%NaCl, 10—HEPES, pH 7.4) containing 900 ppm of2,2-dimethoxy-2-phenoxyacetophenone as initiator, was poured into analuminum mold and irradiated with a LWUV lamp (Black ray) for 1 min. Theinitial weights of these samples were found after oven-drying these gelsto constant weight. The samples were soxhlet-extracted with methylenechloride for 36 hours in order to leach out any unreacted prepolymerfrom the gel matrix (sol-leaching) prior to testing. The process ofextraction was continued until the dried gels gave constant weight.

ICR Swiss male white mice, 6-8 weeks old (Sprague-Dawley), wereanesthetized by an intraperitoneal injection of sodium pentobarbital.The abdominal region of the mouse was shaved and prepared with betadine.A ventral midline incision 10-15 mm long was made. The polymer sample,fully hydrated in sterile PBS (Phosphate buffered saline) or HEPESbuffered saline (for calcification studies), was inserted through theincision and placed over the mesentery, away from the wound site. Theperitoneal wall was closed with a lock stitched running suture (4.0silk, Ethicon). The skin was closed with stainless steel skin staples,and a topical antibiotic (Furacin) was applied over the incision site.Three animals were used for each time point. One dogbone sample wasimplanted per mouse and explanted at the end of 1 week, 3 weeks, 6weeks, and 8 weeks. Explanted gels were rinsed in HBS twice and thentreated with 0.3 mg/mL pronase (Calbiochem) to remove any adherent cellsand tissue. The samples were then oven-dried to a constant weight,extracted, and reswelled as mentioned before.

Tensile stress strain test was conducted on both control (unimplanted)and explanted dogbones in a small horizontal Instron-like device. Thedevice is an aluminum platform consisting of two clamps mounted flat ona wooden board between two parallel aluminum guide. The top clamp wasstationary while the bottom clamp was movable. Both the frictionalsurfaces of the moving clamp and the platform were coated with aluminumbacked Teflon (Cole-Parmer) to minimize frictional resistance. Themoving clamp was fastened to a device capable of applying a graduallyincreasing load. The whole set up was placed horizontally under adissecting microscope (Reichert) and the sample elongation was monitoredusing a video camera. The-image from the camera was acquired by an imageprocessor (Argus-10, Hamamatsu) and sent to a monitor. After breakage, across section of the break surface was cut and the area measured. Theload at break was divided by this cross section to find the maximumtensile stress. Table 8 lists the stress at fracture of PEGtetraacrylate (18.5 kD) hydrogels explanted at various time intervals.No significant change in tensile strength was evident with time. Thus,the gels appear mechanically stable to biodegradation in vivo within themaximum time frame of implant in mice.

TABLE 8 TIME STRESS (KPa) STRAIN AV. IMPLANTED (mean ± error*) (mean ±error*) 1 WK 52.8 ± 16.7 0.32 ± 0.19 3 WK 36.7 ± 10.6 0.37 ± 0.17 6 WK73.3 ± 34.9 0.42 ± 0.26 8 WK 34.1‡ 0.30‡ CONTROL 44.9 ± 5.3 0.22 ± 0.22*Error based on 90% confidence limits. ‡Single sample.

EXAMPLE 22 Monitoring of Calcification of PEO Gels

Disc shaped PEG-tetraacrylate hydrogels (m.w. 18.5 kD) were implantedintraperitoneally in mice as mentioned above for a period of 1 week, 3weeks, 6 weeks, or 8 weeks. Explanted gels were rinsed in HBS twice andtreated with Pronase (Calbiochem) to remove cells and cell debris. Thesamples were then equilibrated in HBS to let free Ca⁺⁺ diffuse out fromthe gel matrix. The gels were then oven-dried (Blue-M) to a constantweight and transferred to Aluminum oxide crucibles (COORS, hightemperature resistant). They wore incinerated in a furnace at 700° C.for at least 16 hours. Crucibles were checked for total incineration, ifany residual remnants or debris was seen they were additionallyincinerated for 12 hours. Subsequently, the crucibles were filled with 2mL of 0.5 M HCl to dissolve Ca⁺⁺ salt and other minerals in the sample.This solution was filtered and analyzed with atomic absorptionspectroscopy (AA) for calcium content.

Calcification data on PEG-tetraacrylate (mol. wt. 18.5 kD) gel implantsis given in Table 9. No significant increase in calcification wasobserved up to an 8 week period of implantation in mice.

TABLE 9 TIME CALCIFICATION (mean ± error*) (Days) (mg Calcium/g of Drygel wt.)  7 2.33 ± 0.20 21  0.88 ± 0.009 42 1.08 ± 0.30 56 1.17 ± 0.26*Error based on 90% confidence limits.

EXAMPLE 23 Encapsulation of Neurotransmitter-releasing Cells

Paralysis agitans, more commonly called Parkinson's disease, ischaracterized by a lack of the neurotransmitter dopamine within thestriatum of the brain. Dopamine secreting cells such as cells from theventral mesencephalon, from neuroblastoid cell lines or from the adrenalmedulla can be encapsulated in a manner similar to that of other cellsmentioned in prior Examples. Cells (including genetically engineeredcells) secreting a precursor for a neurotransmitter, an agonist, aderivative or a mimic of a particular neurotrarsmitter or analogs canalso be encapsulated.

EXAMPLE 24 Encapsulation of Hemoglobin for Synthetic Erthrocytes

Hemoglobin in its free form can be encapsulated in PEG gels and retainedby selection of a PEG chain length and cross-link density, whichprevents diffusion. The diffusion of hemoglobin from the gels may befurther impeded by the use of polyhemoglobin, which is a cross-linkedform of hemoglobin. The polyhemoglobin molecule is too large to diffusefrom the PEG gel. Suitable encapsulation of either native or crosslinkedhemoglobin may be used to manufacture synthetic erythrocytes Theentrapment of hemoglobin in small spheres (<5 μm) of these highlybiocompatible materials would lead to enhanced circulation timesrelative to crosslinked hemoglobin or liposome encapsulated hemoglobin.

Hemoglobin in PBS is mixed with the prepolymer in the followingformulation:

Hemoglobin at the desired amount PEG DA (MW 10000) 35% PEG DA (MW 1000) 5% PBS 60%with 2,2-dimethoxy, 2-phenyl acetophenone at 1.6% of the above solution.

This solution is placed in mineral oil at a ratio of 1 parthemoglobin/prepolymer solution to 5 parts mineral oil and is rapidlyagitated with a motorized mixer to form an emulsion. This emulsion isilluminated with a long-wavelength ultraviolet light (360 nm) for 5 minto crosslink the PEG prepolymer to form a gel. The mw of the prepolymermay be selected to resist the diffusion of the hemoglobin from the gel,with smaller PEG DA molecular weights giving less diffusion. PEG DA ofMW 10000, further crosslinked with PEG DA 1000, should possess theappropriate permselectivity to restrict hemoglobin diffusion, and itshould possess the appropriate biocompatibility to circulate within thebloodstream.

EXAMPLE 25 Entrapment of Enzymes for Correction of Metabolic Disordersand Chemotherapy

Congenital deficiency of the enzyme catalase causes acatalasemia.Immobilization of catalase in PEG gel networks could provide a method ofenzyme replacement to treat this disease. Entrapment of glucosidase cansimilarly be useful in treating Gaucher's disease. Microspherical PEGgels entrapping urease can be used in extracorporeal blood to converturea into ammonia. Enzymes such as asparaginase can degrade amino acidsneeded by tumor cells. Immunogenicity of these enzymes prevents directuse for chemotherapy. Entrapment of such enzymes in immunoprotective PEGgels, however, can support successful chemotherapy. A suitableformulation can be developed for either slow release or no release ofthe enzyme.

Catalase in PBS is mixed-with the pre polymer in the followingformulation:

Catalase at the desired amount PEG DA (MW 10000) 35% PEG DA (MW 1000) 5% PBS 60%with 2,2-dimethoxy, 2-phenyl acetophenone at 1.6% of the above solution.

This solution is placed in mineral oil at a ratio of 1 partcatalase/prepolymer solution to 5 parts mineral oil and is rapidlyagitated with a motorized mixer to form an emulsion. This emulsion isilluminated with a long-wavelength ultraviolet light (360 nm) for 5 minto crosslink the PEG prepolymer to form a gel. The mw of the prepolymermay be selected to resist the diffusion of the catalase from the gel,with smaller PEG DA molecular weights giving less diffusion.

PEG DA of MW 10,000, further crosslinked with PEG DA 1000, shouldpossess the appropriate permselectivity to restrict catalase diffusion,and it should possess the appropriate permselectivity to permit thediffusion of hydrogen peroxide into the gel-entrapped catalase to allowthe enzymatic removal of the hydrogen peroxide from the bloodstream.Furthermore, it should possess the appropriate biocompatibility tocirculate within the bloodstream.

In this way, the gel is used for the controlled containment of abioactive agent within the body. The active agent (enzyme) is large andis retained within the gel, and the agent upon which it acts (substrate)is small and can diffuse into the enzyme rich compartment. However, theactive agent is prohibited from leaving the body or targeted bodycompartment because it cannot diffuse out of the gel compartment.

EXAMPLE 26 Cellular Microencapsulation for Evaluation of Anti-humanImmunodeficiency Virus Drugs in vivo

HIV infected or uninfected human T-lymphoblastoid cells can beencapsulated into PEG gels as described for other cells above. Thesemicrocapsules can be implanted in a nonhuman animal and then treatedwith test drugs such as AZT or DDI. After treatment, the microcapsulescan be harvested and the encapsulated cells screened for viability andfunctional normalcy using a fluorescein diacetate/ethidium bromidelive/dead assay. Survival of infected cells indicates successful actionof the drug. Lack of biocompatibility is a documented problem in thisapproach to drug evaluations, but the highly biocompatible gelsdescribed herein solve this problem.

EXAMPLE 27 Use of PEG Gels as Adhesive to Rejoin Severed Nerve

A formulation of PEG tetraacrylate (10%, 18.5 kD), was used as adhesivefor stabilizing the sutureless apposition of the ends of transectedsciatic nerves in the rat. Rats wore under pentobarbital anesthesiaduring sterile surgical procedures. The sciatic nerve was exposedthrough a lateral approach by deflecting the heads of the bicepsfemoralis at the mid-thigh level. The sciatic nerve was mobilized forapproximately 1 cm and transected with iridectomy scissors approximately3 mm proximal to the tibeal-peroneal bifurcation. The gap between theends of the severed nerves was 2-3 mm. The wound was irrigated withsaline and lightly swabbed to remove excess saline. Sterile,unpolymerized PEG tatraacrylate solution was applied to the wound. Usingdelicate forceps to hold the adventitia or perineurium, the nerve endswere brought into apposition, the macromer solution containing2,2-dimethoxy-2-phenylacetophenone as a photoinitiator and the wound wasexposed to long wavelength UV-light (365 nm) for about 10 sec topolymerize the adhesive. The forceps were gently pulled away. Care wastaken to prevent the macromer solution from flowing between the twonerve stumps. Alternatively, the nerve stump junction was shielded fromillumination, e.g. with a metal foil, to prevent gelation of themacromer solution between the stumps; the remaining macromer solutionwas then simply washed away.

In an alternative approach, both ends of the transected nerve were heldtogether with one pair of forceps. Forcep tips were coated lightly withpetrolatum to prevent reaction with the adhesive. The polymerizedadhesive serves to encapsulate the wound and adhere the nerve to theunderlying muscle. The anastomosis of the nerve ends resists gentlemobilization of the joint, demonstrating a moderate degree ofstabilization. The muscle and skin were closed with sutures.Reexamination after one month shows that severed nerves can remainreconnected, despite unrestrained activity of the animals.

EXAMPLE 28 Surgical Adhesive

The unpolymerized macromer mixture was an aqueous solution, such as thatof PEO 400 kD diacrylate or PEO 18.5 kD tatraacrylate. When thissolution contacts tissue, which has a moist layer of mucous or fluidcovering it, it intermixes with the moisture on the tissue. The mucouslayer on tissue comprises water soluble polysaccharides, whichintimately contact cellular surfaces. These, in turn, are rich inglycoproteins and proteoglycans. Thus, physical intermixing, hydrogenbonding, and forces of surface interlocking due to penetration intocrevices are some of the forces responsible for the adhesion of the PEOgel to a tissue surface subsequent to crosslinking.

PEO diacrylate solutions can therefore be used as tissue adhesives as inthe previous example. Specific applications for such adhesives mayinclude blood vessel anastomosis, soft tissue reconnection, drainableburn dressings, and retinal reattachment. However, if the PEO gel ispolymerized away from tissue, it then presents a very non-adhesivesurface to cells and tissue in general, due to the low interfacialenergy intrinsic to the material.

Abdominal muscle flaps from female New Zealand white rabbits wereexcised and cut into strips 1 cm×5 cm. The flaps were approximately 0.5to 0.8 cm thick. A lap joint (1 cm×1 cm) was made using two such flaps.Two different PEO di- and (tetra-) acrylate macromer compositions, 0.4kD (di-) and 18.5 kD (tetra-), were evaluated. The 0.4 kD compositionwas a viscous liquid and was used without further dilution. The 18.5 kDcomposition was used as a 23% w/w solution in HBS. 125 μL of ethyl eosinsolution in n-vinyl pyrrolidone (20 mg/mL) along with 50 μL oftriethanolamine was added to each mL of the adhesive solution. 100 μL ofadhesive solution was applied to each of the overlapping flaps. The lapjoint was then irradiated by scanning with a 2 W argon ion laser for 30seconds from each side. The strength of the resulting joints wasevaluated by measuring the force required to shear the lap joint. Oneend of the lap joint was clamped and an increasing load was applied tothe other end, while holding the joint horizontally until it failed.Four joints were tested for each composition. The 0.4 kD joints had astrength of 12.0 ± 6.9 KPa (mean±S.D.), while the 18.5 kD joints had astrength of 2.7±0.5 KPa. It is significant to note that it was possibleto achieve photopolymerization and reasonable joint strength despite the6-8 mm thickness of tissue. A spectrophotometric estimate using 514 nmlight showed less than 1% transmission through such muscle tissue.

EXAMPLE 29 Modification of PVA Polymer

Water soluble polymers other than PEG can also be modified using asimilar chemical approach to that in prior examples. Thus, poly(vinylalcohol) (PVA), which is water soluble, can be easily crosslinked togive a gel wherein the crosslinking occurs under conditions mild enoughto permit gelation in contact with or in the vicinity of biologicalmaterials such as tissues, mammalian cells, proteins, orpolysaccharides. The method adopted herein leads to PVA crosslinkingunder very mild conditions as compared to the standard techniques offormaldehyde crosslinking.

Polyvinyl alcohol (2 g; m.w. 100,000-110,000 D) was dissolved in 20 mLof hot DMSO. The solution was cooled to room temperature, and 0.2 mL oftriethylamine and 0.2 mL of acryloyl chloride was added with vigorousstirring under an argon atmosphere. The reaction mixture was heated to70° C. for 2 hr and cooled. The polymer was precipitated in acetone,redissolved in hot water, and precipitated again in acetone. Finally, itwas dried under vacuum for 12 hr at 60° C. A solution of this polymer inPBS (5-10% w/v) was mixed with the UV photoinitiator and polymerizedusing long wavelength UV light to make microspheres 200-1,000 microns insize. These microspheres wore stable to autoclaving in water, whichindicates that the gel was covalently cross-linked. The gel wasextremely elastic. This macromer, PVA multiacrylate, may be used toincrease the crosslinking density in PEG diacrylate gels, withcorresponding changes in mechanical and permeability properties. Thisapproach could be pursued with any number of water-soluble polymerswhich are chemically modified with photopolymerizable groups; forexample, with water-soluble polymers chosen from polyvinylpyrrolidone,polyethyloxazoline, polyethyloneoxide-polypropyleneoxide copolymers,polysaccharides such as dextran, alginate, hyaluronic acid, chondroitinsulfate, heparin, heparin sulfate, heparan sulfate, guar gum, gel Iangum, xanthan gum, carrageenan gum, etc., and proteins such as albumin,collagen, gelatin, and the like.

EXAMPLE 30 Use of Alternative Photopolymerizable Moieties

Many photopolymerizable groups may be used to enable gelation. Severalpossibilities are shown below, based upon a PEG central chain:

where F₁=CONH, COO or NHCOO

-   -   X=H, CH₃, C₂H₅, C₆H₅, Cl, Br, OH or CH₂COOH    -   F₂=COO, CONH, O or C₆H₄,    -   R=CH₂ or -alkyl-,    -   n 5, and    -   m 3.

To illustrate a typical alternative synthesis, a synthesis for PEG 1 kDurethane methacrylate is described as follows, where F₁=NHCOO, F₂=COO,R=CH₃ and X=CH₃:

In a 250 mL round bottom flask, 10 g of PEG 1 kD was dissolved in 150 mLbenzene. 2-isocyanatoethylmethacrylate (3.38 g) and 20 pL ofdibutyltindilaurate were slowly introduced into the flask. The reactionwas refluxed for 6 hours, cooled, and poured into 1000 mL hexane. Theprecipitate was filtered and dried under vacuum at 60° C. for 24 hours.

EXAMPLE 31 Use of Alternative Photoinitiator/Photosensitizer Systems

It is possible to initiate photopolymerization with a wide variety ofdyes as initiators and a number of electron donors as effectivecocatalysts. Table 10 illustrates photopolymerization initiated byseveral other dyes, which have chromophores absorbing at widelydifferent wavelengths. All gelations were carried out using a 23% w/wsolution of 18.5 kD PEG tetraacrylate in HEPES buffered saline. Theseinitiating systems compare favorably with conventional thermalinitiating systems, as can also be seen from Table 10.

TABLE 10 Polymerization Initiation LIGHT TEMPERATURE APPROXIMATEINITIATOR SOURCE* ° C. GEL TIME, (SEC) Eosin Y, 0.00015M; S1 with UV 2510 Triethanolamine 0.65M filter Eosin Y, 000015M; S4 25 0.1Triethanolamine 0.65M Methylene Blue, 0.00024M; S3 25 120p-toluenesulfonic acid, 0.0048M 2,2-dimethoxy-2-phenyl S2 25 8aceto-phenone 900 ppm Potassium Persulfate 0.0168M — 75 180 PotassiumPersulfate 0.0168M; — 25 120 tetramethyl ethylene-diamine 0.039MTetramethyl ethylene-diamine S1 with UV 25 300 0.039M; Riboflavin0.00047M filter *LIST OF LIGHT SOURCES USED CODE SOURCE S1 Mercury lamp,LEITZ WETZLER Type 307-148.002, 100W S2 Black Ray longwave UV lamp,model B-100A W/FLOOD S3 MELLES GRIOT He--Ne laser, 10 mW output, 1 = 632nm S4 American laser corporation, argon ion laser, model 909BP-15-01001;1 = 488 and 514 nm

Numerous other dyes can be used for photopolymerization. These dyesinclude but are not limited to Erythrosin, phloxime, rose bengal,thionine, camphorquinone, ethyl eosin, eosin, methylene bluer andriboflavin. Possible cocatalysts that can be used include but are notlimited to: N-methyl diethanolamine, N,N-dimethyl benzylamine,triethanolamnine, triethyl amine, dibenzyl amine, N-benzyl ethanolamine,N-isopropyl benzylamine.

EXAMPLE 32 Formation of Alginate-PLL-alginate Microcapsules WithPhotopolymerizable Polycations

Alginate-polylysine-alginate microcapsules were made by adsorbing, orcoacervating, a polycation, such as polylysine (PLL), upon a gelledmicrosphere of alginate. The resulting membrane was held together bycharge-charge interactions and thus has limited stability. To increasethis stability, the polycation can be made photopolymerizable by theaddition of carbon—carbon double bonds, for example. This can be used toincrease the stability of the membrane by itself, or to react, forexample, with photopolymerizable PEG to enhance biocompatibility.

To illustrate the synthesis of such a photopolymerizable polycation, 1 gof polyallylamine hydrochloride was weighed in 100 mL glass beaker anddissolved in 10 mL distilled water (DW). The pH of the polymer solutionwas adjusted to 7 using 0.2 M sodium hydroxide solution. The polymer wasthen separated by precipitating in a large excess of acetone. It wasthen redissolved in 10 mL DW and the solution was transferred to 50 mLround bottom flask. Glycidyl methacrylate (0.2 mL) was slowly introducedinto the reaction flask and the reaction mixture was stirred for 48hours at room temperature. The solution was poured into 200 mL acetoneand the precipitate was separated by filtration and dried in vacuum.

In addition to use in encapsulating cells in materials such as alginate,such photopolymerizable polycations may be useful as a primer orcoupling agent to increase polymer adhesion to cells, cell aggregates,tissues, and synthetic materials, by virtue of adsorption of thephotopolymerizable polymer bonding to the PEG photopolymerizable gel.

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1. A method for encapsulation of at least one islet cell encapsulated ina microcapsule, comprising the steps of: a) coating at least one isletcell encapsulated in a microcapsule with photoinitiator; b) suspendingthe at least one coated islet cell encapsulated in a microcapsule in amacromer solution comprised of macromer; and c) irradiating thesuspension with light.
 2. The method of claim 1, wherein the macromer isa water soluble, ethylenically unsaturated, polymer susceptible topolymerization into water insoluble polymer through interaction of atleast two carbon-carbon double bonds.
 3. The method of claim 2, whereinthe macromer is selected from the group consisting of ethylenicallyunsaturated derivatives of poly(ethylene oxide) (PEO), poly(ethlyeneglycol) (PEG), poly(vinyl alcohol) (PVA), poly(vinylpyrrolidone) (PVP),poly(ethyloxazoline) (PEOX), poly(amino acids), polysaccharides, andproteins.
 4. The method of claim 3, wherein the polysaccharides areselected from the group consisting of alginate, hyaluronic acid,chondroitin sulfate, dextran, dextran sulfate, heparin, heparin sulfate,heparan sulfate, chitosan, gellan gum, xanthan gum, guar gum, watersoluble cellulose derivatives and carrageenan.
 5. The method of claim 3,wherein the proteins are selected from the group consisting of gelatin,collagen, and albumin.
 6. The method of claim 1, wherein thephotoinitiator is any dye that absorbs light having a frequency between320 nm and 900 nm, can form free radicals, is at least partially watersoluble, and is non-toxic to the at least one islet cell at theconcentration used for polymerization.
 7. The method of claim 1, whereinthe macromer solution further comprises a primary, secondary, tertiary,or quaternary amine cocatalyst and the photoinitiator is selected fromthe group of ethyl eosin, eosin Y, fluorescein, 2,2-dimethoxy,2-phenylacetophenone, 2-methyl, 2-phenylacetonphenone, camphorquinone,rose bengal, methylene blue, erythosin, phloxime, thionine, riboflavin,and methyl green.
 8. The method of claim 1, wherein the microcapsule iscomprised of material selected from the group of alginate, chitosan,agarose, and gelatin.
 9. The method of claim 1, wherein the macromersolution further comprises an accelerator to increase the rate ofpolymerization.